We have recently discovered that human cells and tissues synthesize three forms of cytoplasmic dynein. The dynein heavy chains (DHCs) in these enzymes are encoded by three distinct genes. lsotype-specific antibodies have been raised against polypeptides expressed in bacteria from recombinant fragments of each DHC gene. Both these and gene-specific probes reveal different levels of expression in each cell studied. The three dyneins have interestingly different localizations within cells. DHC1, the form already studied in HeLa cells and rodent brain, is in the mitotic spindle, with concentrations on the centrosomes and kinetochores. During interphase, it is associated with cytoplasmic vesicles and perhaps with primary endosomes. DHC2 is concentrated in the Golgi apparatus and on cytoplasmic vesicles. DHC3 is associated with a reticular or stringy cytoplasmic structure during interphase, which may be a subcompartment of the ER or the compartment intermediate between ER and Golgi. We propose to study the functions of these dynein isoforms in vivo and in vitro. Immunological and molecular methods will be used to perturb their actions in living cells, and the resulting phenotypes will be studied by microscopy and a variety of physiological assays that monitor membrane traffic and behavior in vivo. We will purify the holo-enzymes that include each of these isoforms, identify any lower molecular weight polypeptides with which each DHC associates, and characterize any ATPase activities, motor actions on microtubules, and interactions with potential regulatory complexes, like dynactin. We will work with in vitro systems that reconstitute some aspect of.chromosome motion or membrane traffic, so the roles of each dynein isoform can be explored under well controlled experimental circumstances. A laser trap will be used, together with stable or labile microtubules, to control the framework over which different populations of vesicles or chromosomes can move, and the roles of dynein in these movements will be assessed by studying the effects of added enzyme, function blocking antibodies and/or recombinant fragments of the DHCs that might compete for essential binding activities and block the action of the holo-enzymes. Through these experiments we hope to learn the roles played by each isoform of cytoplasmic dynein in cell growth and physiology. Finally, we will improve our knowledge of the gene structures and map positions of each DHC to look for related disease genes or mutations that might help us understand these enzymes, and we will seek additional isoforms of cytoplasmic DHCs.